Vascular inflammation is recognized as a critical process in the initiation, progression and complications of insulin resistance, type 2 diabetes, and atherosclerosis. Toll-like receptors (TLRs) are traditionally described as cellular sensors for microbial pathogens, but more recently demonstrated by us and others to recognize modified (e.g. oxidized) host molecules, leading to TLR-mediated inflammatory responses by vascular cells. Human epidemiologic and animal experimental studies have implicated TLR4 in the development of insulin resistance, diabetes and atherosclerosis. Work from our lab has shown that in macrophages, TLR4 signaling is activated by minimally oxidized LDL (mmLDL), an early form of oxidized LDL found in atherosclerotic lesions. In addition, TLR4 is activated by lipopolysaccharide (LPS), the prototypical bacterial endotoxin, and emerging evidence suggests that persistent subclinical endotoxemia is an integral component of metabolic disorders induced by Western type, high-fat diets, and has been termed metabolic endotoxemia. We have demonstrated that injections of low doses of LPS and mmLDL cooperatively (and even synergistically) activate macrophages in a TLR4-dependent manner to express higher levels of proinflammatory cytokines. In this grant proposal, we will test the hypothesis that metabolic endotoxemia, induced by a high-fat diet, together with oxidized lipids, components of mmLDL, synergistically enhance vascular inflammation. These experiments will help elucidate mechanisms of accelerated atherosclerosis in obese and diabetic patients. Specifically, we will study oxidized cholesteryl esters (OxCE), produced in LDL as a result of oxidative modification by 12/15-lipoxygenase (12/15LO). 12/15LO is a major enzyme that promotes LDL oxidation In vivo, and has been implicated in the onset of adipose tissue inflammation and insulin resistance, development of diabetic vasculopathy and atherosclerosis. We have identified CE oxidized via 12/15LO catalysis as active components of mmLDL responsible for TLR4-dependent proinflammatory effects in macrophages. Importantly, we have shown the presence of such OxCE in murine atherosclerotic lesions. In this application, we will study In vivo vascular inflammation induced by cooperative stimulation with mmLDL, OxCE and endogenous 12/15LO activity, on the one hand, and metabolic endotoxemia on the other. Moreover, we will utilize mass spectrometry techniques and OxCE-specific antibodies to provide evidence for the importance of OxCE in pathophysiological processes, and for development of novel biomarker and imaging applications.